Q&A for all webinars

Harold McNair, Virginia Tech, USA

LevelBasic
The First webinar in the year of education was held by Harold McNair: Introduction to Gas Chromatography. We decided to start with this really basic seminar because we anticipated a large demand on these basics. And we were right, over 900 attended the webinar. The live webinar has been recorded and can be viewed here.

"Introduction to GC" has also been recorded as a video presentation. Click here to go to the series of five, click here to watch a free taster.

During the webinar we received a large number of questions. Professor Harold McNair has answered them. You will find the questions and answers below:

Introduction to Gas Chromatography

     “Introduction to Gas Chromatography” Webinar
                 28 October 2009
            Question and Answers

1. For FID could I consider any area percent as concentration in wt %?	No.  Area percent is not weight percent.  You need to make a calibration curve with pure standards.  A good example is for ECD; 1µg of benzene, toluene and xylene might generate  peak areas of 1000, 3000 and 5000.You must calibrate for each compound.
2. With sample cleanup, I doubt that the quantitative aspects are guarantueed?	If you add an internal standard to the sample matrix before clean-up you will normally get better quantitative results.
3. How is Gas chromatography going to deal the eventual exhaustion of Helium?  Will Hydrogen become the standard carrier gas in the future?  When do we run out of Helium?	No good answer at this time.  Hydrogen will be used more as carrier gas, however helium will remain the most widely used for some time.
4. My lab uses hydrogen as a carrier gas and for GC-MS and GC-FID instead of helium. Considering the flammability of hydrogen, as well as the expense, are there advantages to using hydrogen that outweigh these issues?	I see no practical advantage in using H2 over He today.
5. What is the use /function of the wool in the injection port?	Silanized glass wool is used primarily to adsorb non-volatile components in dirty samples.  It also provides a hot, large surface area to help vaporize volatiles.
6. What is the min. quantity (In Grams) for the Sample?	For capillary columns, typical sample concentrations range from nanograms up to 100 micrograms in a solvent. Typical injection volumns are 1 microliter.
7. Hallo - i want to know how can i make a quantification analysis by using SPME GC-MS injection	Too complicated to be answered here. Please check the Chromedia topics on quantitative analysis, SPME and GC-MS
8.  Is there any reason why Hydrogen gas cannot be used as carrier gas when using TCD, even we are not interested in analyzing Hydrogen??	Hydrogen can be used with the TCD.  It cannot be used to measure hydrogen or helium.
9.  Please define column capacity.	A broader than normal peak , (I call it cigar shaped) is a good indication of an over-loaded detector or column.  When retention time decreases with larger samples it usually indicates  an overloaded column.
10. How can i calculate the LOD of GC-MS by using calibration series	First measure the noise  level.  Then make up a series of pure standards, maybe 10ppm, 1ppm, 0.1ppm etc.  When the peak area is 10X noise level, that is the LOQ, limit of quantization.  When the peak area is 3X noise level, that is the LOD, limit of detection.  That peak is present in the sample, but you cannot report any valid concentration.
11.  What can we do with compounds with high molecular weight and not volatile ? Is their is any CG coupled with protone NMR,13-C NMR and mass?	If truly non-volatile, there is nothing you can do.  Molecules have to be in the vapor state to be carried through a GC column.
12.  What about  the column unions for capillary?	Yes, use a zero dead volume fitting, or  end to end fitting of identical I.D.s.
13.  The detection limit of GC can be as low as picogram.  Is "picogram" meant the amount injected into GC?	No, the detection limit, LOD is the smallest amount of any component in the sample which can be detected. Please check Chromedia and textbooks on this topic.
14.  What is the advantage of split injection system - Prof Deokate U.A.	As capillary columns demand a very small amount of sample injected rapidly, a split injections meets both demands.  It is also very simple, open a split vent.
15.  What is the cause of seeing series of siloxane peaks in my blanks example "Cyclodecasiloxane, eicosamethyl-". Is it a bleeding column or liner problem?	Siloxane peaks can come from both the septum and the column liquid phase.  Use a faster septum purge (4ml/min) or a Teflon backed septum to minimize septum “bleed.”  Since column bleed is proportional to the amount of liquid phase:  use a shorter column, a thinner film, or a more temperature stable liquid phase.  If possible use a lower column temperature.
16.  Can  sample be vaporized at sample point or in GC oven rather than hot injection port?	Sample can be vaporized in a heated inlet liner, or inside the column itself (on-column injector or PTV).
17.  Can you explain splitless and split/splitless injection?	Too complicated for here, I refer to Chromedia, which has sections on these topics.
18.  What would you recommend to replace glass wool in the SSL liner due to possible adverse activity when dealing with dirty samples?	Be sure to use “silanized” glass wool, and replace it frequently.
19.  Powder or bigger chip (>1.00 mm) which one is Better?	I do not understand the question.
20.  Hello, about the sampling of very volatile samples especially iso-pentane. we introduce the sample as liquid but we loose the sample at the time of injection. we got very poor peaks. thank you	Use a gas sample valve; add a restrictor to the exit end of the valve to keep the gas sample loop  under high pressure, at least 2-4psi.This should keep the pentane in the liquid state.
21.  What do you think is the future for GC?  There are some interesting news	The future of GC is slow evolution, no more revolution( my opinion).  We have worked on improving GCs for over 50 years.  The columns, detectors and data systems are almost at the optima.  We will see faster GC, more GC/MS and emphasis on automated sample handling.
22.  Can you touch upon the difference between a PTV injection port and the S-SL injection port?	Sorry, this question is beyond my expertise.  Chromedia will ask within their expert team.
23.  How maintenance I mean cleaning is given to a ECD  after time	Maintain the ECD at a high temperature to avoid condensation of sample or column “bleed.”  Annually clean the ECD by passing H2 as carrier gas through the hot detector overnight.  Use either an empty column, or a solid phase column like silica gel which does not bleed.  The hot H2 will burn off any organic residue on the radioactive foil.
24.  Which gives better resolution, thick or thin film?	Thin films give better resolution than thick films.  Please refer to the Golay Equation for capillary columns.
25.  For trace analysis, what are the  advantages and disadvantages of using large volume injection?	Large volume injection has the advantage of better detection limits.  Its disadvantage is that usually it involves a special accessory, that is extra cost and inconvenience.
26.  I am carrying out a chemical reaction in solution  (with dry organic solvent 20 mL) in a flask with inert atmosphere. I suspect that the reaction releases molecular hydrogen gas, however bubbles are not seen. I think that H2 (about 0.1 mmol) is dissolved in the solvent. Could GC technique detect this H2?	H2 can easily be detected by GC, but not with a FID.  You will need to use a TCD or MS.
27.  How do you coat tubing walls with liquid phase? How do you keep the liquid phase stationary?	Making capillary columns is too complicated for the time available.  Please refer to a good textbook.  “Modern Practice of GC” by Grob and Berry, John Wiley 2004.
28.  Do you need to split injections with a megabore column?	Some injections can be made without splitting on a thick film, megabore column, but you need to start at a low temperature and then temperature programs the column.  A split injection will always give better resolutionon all capillary columns .
29. When do you using constant flow and constant pressure mode ?	Use constant pressure only with iso-thermal separations, for all other methods, including iso-thermal use constant flow.We use constant flow in all situations
30. How would you trouble shoot a signal value that increases during a temperature increase.  This looks like the baseline has shot up during the final temperature stage.	Several options here. Could be column bleeding. Please check the troubleshooting section on Chromedia.

Sample prep

Questions and Answers – Chromedia April 2010

Nicholas H. Snow, Seton Hall University

How would you remove natural organic matter (NOM; like humic and fulvic acids) in water samples? I am extracting 500 ml of source water.

Think about the solubility of the matrix components as well as the analytes. If using an SPE-based method, you might consider pH adjustment to deprotonate the acids (high pH) so they stay dissolved in the water as is passes through the SPE cartridge or is mixed with an oragnic solvent. You want to plan an extraction where the analytes are soluble in the extraction phase but the interferences are not. Check application notes and literature from SPE vendors.

Is SPE a good method for extracting proteins, for example samples as milk, vaccines?

Yes. Best next step would be to check application notes at one of the major vendors for SPE materials and supplies.

Are all SPE C18 cartridges the same (different vendors)?

No. Each vendor will use a different source for the particles themselves and a different procedure for preparing the bonded phase. Like HPLC columns, they have subtle differences in properties, so you will need to perform experiments to determine equivalence.

"When do you have a complex soil matrix, what type of extraction of organic pollution is more useful for this purpose?"

There are several possibilities, depending on how strongly you suspect the organic contaminants are bound to the soil – for instance were they spilled today or have they been seeping into the soil for years? Listed in order of simple and inexpensive to more challenging and expensive, I would explore.

1. Try extracting with an appropriate organic solvent. Use an ultrasonic bath to assist if needed.
2. Soxhlet extraction.
3. Accelerated Solvent Extraction
4. Supercritical Fluid Extraction

Definitely check the literature and think about the goals of your analysis before beginning.

What is a matrix? (in slide 12). Can we use this technique for quantitative separation?

The matrix is all that stuff in your sample that is not the compounds of interest. You can use all of the discussed techniques for quantitative analysis. For multi-step techniques such as SPE, SPME, ASE, etc., I suggest you plan to use an internal standard, added to all samples, blanks and standards prior to extraction, to account for variable extraction recovery.

How do you begin to develop an SPE separation method, when there are so many cartridges, solvents, and individual component properties? How do you validate your separation protocol?

I begin by thinking about he solubility of my analytes and major matrix components. We are looking for a sorbent and solvent combination that will initially trap both analytes and matrix on the sorbent, then allow washing of the matrix from the sorbent while retaining the analytes, then allow elution of the analytes. Nearly all of the major vendors of SPE materials have method development guides and examples on their websites; this is a good place to begin.

In your experience, have you found SPE vendors to be variable in their sorbents: both vendor-to-vendor as well as lot-to-lot variability from a single vendor?

Yes. This is one reason that I generally use internal standard quantitation with SPE. Recovery from lot to lot and vendor to vendor may vary significantly. Also there is often analyst to analyst variability as well.

What happens if you have a family of compounds with very similar properties and you need to separate only one of them that is in very low concentration, How can you handle this task?

In general, I would think of extraction as a bulk phase transfer process. Separate my analyte and the similar compounds from all the rest of the matrix (like all the steroids from urine) using the extraction method. Next use high resolution gas or liquid chromatography to separate the compounds in the family from each other. Extraction methods alone generally lack the selectivity to separate closely related compounds.

bulk C18 or prepacked C18 cartridges?

For SPE, premade cartridges will be much easier to use and more reproducible.

How critical is the extraction time in SPME and SBSE? Is the time important to reproducibility?

Extraction time is critical in all extraction methods. During method development, it is important to optimize extraction time be performing several experiments using different extraction times and plotting peak area versus extraction time. As time increases, the plot should increase until it reaches a plateau. Extraction time should be set to a time reflected on the plateau so that small variations in time will not have a major effect on peak area.

I am extracting multi water soluble vitamins from fish oil. What approach do you suggest with SPE. Currently we are using hexane extraction. With no recovery studies done, I would like to move to SPE extraction.


How does the solvent drop stay on the end of the syringe in that last micro-extraction technique?

In single drop micro-extraction a drop of organic solvent is suspended from the tip of a micro-syringe. The drop stays on the tip because the surface tension of the solvent and its repulsion of water will help keep it intact. Of course, with too large a drop or too much agitation, the drop may fall off.

Do you have any suggestions or strategies regarding desorption from adjuvants (e.g. alum salts) of recombinant proteins particularly in multivalent vaccine preparations (e.g. 3 or more proteins)? Thanks very much!


When you use the SPME you are only extracting the analytes and getting rid of solvent. Is it any solvent, for example would it work for dodecane as a solvent ?

Just like liquid-liquid extraction, you should be sure that the sample solvent is not soluble in the fiber material. In the case of dodecane as analyte solvent, dodecane would likely overload the fiber.

Follow up question--- If quantitating with an internal standard with SPE, what are the requirements for internal standard , eg, can we use same internal standards we used with GC?

You can generally use the same internal standards you would use for a GC method. Similar GC properties will most likely lead to similar SPE properties. For GCMS you can often use deuterated analogs of your analytes as internal standards.

Can you address how to obtain a representative sample or sub-sample for analysis?

This is one of the biggest challenges and easiest things to challenge in any analytical method. For a sample to be representative, it must be taken from a homogeneous mixture that is also at equilibrium. Assuring these two conditions is often difficult. When sampling, I note carefully any observations that may lead to doubt about whether the sample is representative.

How many samples (or a range) are usually measured to validate a method ?

The number of required samples, blanks and standards required to validate a method will vary among industries and applications. In general, you need enough samples to generate valid statistics for:
Precision – how close repeated measurements of the same sample are to each other
Accuracy – how close a measurement is to the true value
Detection limit – the smallest amount of analyte that can be detected
Sensitivity – how much does the instrument response change if the amount of analyte changes
The specific procedures for measuring these will vary between industries. Guidelines are generally promulgated by regulatory bodies or industry groups such as USP, ICH, ASTM, AOAC, EPA, etc.

What is spme?

SPME stands for solid phase micro-extraction. There is an excellent discussion in the sample preparation section of chromedia.org.

Would you introduce again the book you mentioned earlier?

There are numerous excellent books and introductory materials related to sample preparation. A few of my favorites are:

1. www.Chromedia.org. SPME section.
2. Pawliszyn, Solid Phase Microextraction Theory and Practice, Wiley, 1997.
3. Kolb and Ettre, Static Headspace Gas Chromatography, 2nd. Edition, Wiley, 2006.
4. sample preparation chapters in Grob, Modern Practice of Gas Chromatography, 4th. Edition, Wiley, 2003 and Miller and McNair, Basic Gas Chromatography, 2nd. Edition, Wiley, 2009.

How does work in detail single drop extraction ? Do you need a special type of syringe?

No special syringe needed – a standard pointed needle GC syringe will do. You will want to place your sample into a vial. Then draw your extraction solvent into the syringe and insert the needle containing the solvent into the sample liquid. Depress the plunger slowly to form the solvent drop in the sample. Suspend to complete extraction then withdraw the extraction solvent into the needle and transfer to the GC for injection.

Any experience/thoughts/ futher references on FBE (fluid bed extraction)?

Fluidized bed extraction is simply solid phase extraction without the cartridge. The solid phase material is placed directly into the sample and agitated as in a reaction. The solid phase material and extracted analytes are then recovered and the analytes are desorbed by combining with an elution solvent. The advantages are control over the amount and type of sorbent and it is quite simple. Disadvantages are that it is labor intensive.

On the methods such as stir bar or single drop, what is the accuracy of quantitation?

In SBSE and SPME quantitation is usually very accurate with results generally within a few percent of the true value for standards. Precision may vary from a few percent to +/- 10-20%, depending on extraction conditions and sample matrix. For best precision, extraction time, temperature and agitation rate should be carefully controlled. Generally, I use internal standard quantitation with both techniques.

Any comments on "Disposable Pipette EXtraction (DPX)"? Thanks.

Disposable pipette extraction is exactly as the name implies. Solid phase extraction sorbent is placed in a disposable pipette tip and extraction is formed by drawing sample, followed by wash solvent followed by elution solvent through it. The sorbent is retained in the disposable pipette tip and discarded following extraction. It should work in a similar manner to SPE, except that usually a smaller mass of sorbent is used.

You did not discuss the importance of MAE? How You rate MAE in analysis as compare to other sample prep techniques?

This was an omission on my part (sorry). Microwave Assisted Extraction is also useful for extracting analytes from solids or other difficult matrices. It is very efficient in terms of energy transfer into the sample matrix.

Explain SPME and pesticide extraction slide. You skipped over it. Usually, pest ext involves a lot of organic solvents - bad!

This was simply the extraction of a standard mixture form water. SPME is potentially good for pesticides because it reduces exposure to reactive surfaces such as glassware and filter materials. A literature reference is provided on the slide for all the details.

What do you think about QUECHERS ? What do these kits cost ?

QUECHERS stands for “quick, easy, cheap, effective, rugged and safe” analysis for pesticide residues, especially in combination with GCMS and LCMS. Im short, a homogenized sample is combined with appropriate solvent(s) and passed through a SPE cartridge for cleanup prior to GC and LC analysis. An online search will turn up numerous applications. QUECHERS method is reported to be less expensive than traditional SPE.

Do you have experience with SFE (supercritical fluid extraction)?

Yes. SFE generally involved the use of supercritical carbon dioxide as extraction solvent and is hardware intensive as it operates at elevated temperatures and pressures. For a very basic introduction see: N.H. Snow, M. Dunn and S. Patel, “Determination of Crude Fat in Food Products by Supercritical Fluid Extraction and Gravimetric Analysis,” Journal of Chemical Education, 1997, 74(9), 1108-1111

If so, how can you fractionate what is extracted?

In SFE, the polarity of the fluid can be adjusted by mixing it with an organic modifier. Fractionation can occur by collecting separate fractions as the elute from the extraction cartridge and as the solvent polarity is changed.

When is best to use accelerated solvent extraction?

ASE has its strength in extracting compounds that are readily soluble in traditional solvents from solids.

What is more convenient? Use more times an low volume or less times and more volume during the extraction?

A single large extraction will always be simpler. Multiple extractions using smaller volumes that add up to the larger volume will produce a larger recovery of a more pure extract. Multiple extractions are also favorable in cases where the partition coefficient favors the original (sample) phase. See the sample preparation chapter in Grob, Modern Practice of Gas Chromatography, 4th. Edition, Wiley, 2003, for more details.